An important question concerning signaling by the insulin-like growth factor I (IGF-I) receptor is whether the downstream effector protein SHC interacts directly with the receptor or with a docking protein called insulin receptor substrate 1 (IRS-1). In collaboration with Dr. Richard Furlanetto, University of Rochester, we have employed the yeast two hybrid system to explore possible direct interactions between the IGF-I receptor and SHC. This utilizes a hybrid protein containing the bacterial LexA DNA binding domain fused to the intracellular portion of the IGF-I receptor, hybrids containing an activation domain fused to full length SHC or fragments of SHC, and a yeast strain containing LexA operator sequences fused to leu and lac Z reporter genes. When yeast were transfected with the LexA-IGF-I receptor plasmid, a fusion protein of the predicted molecular size was detected by Western blotting. This protein contained phosphotyrosine indicating that the receptor had undergone autophosphorylation. Coexpression of LexA-IGF-I receptor with activation domain-SHC hybrid resulted in activation of both reporter genes. Neither reporter gene was activated when an IGF-I receptor kinase negative mutant was used in the two-hybrid system. Surprisingly, coexpression of LexA-IGF-I receptor hybrid with activation domain SHC-SH2 domain hybrid resulted in much weaker interaction than obtained with full length SHC. This result tells us that a domain of the SHC molecule outside of the SH2 domain was largely responsible for the strong interaction of the receptor and full length SHC, and is consistent with recent reports describing interaction of the amino terminal portion of SHC with phosphotyrosine containing proteins. Studies have now been completed that test the hypothesis that endogenous IGF-II can modulate the trafficking of lysosomal enzymes by blocking binding to the IGF- II/mannose 6-phosphate receptor. MCF-7 human breast cancer cells overexpressing a precursor form of IGF-II exhibit a decreased ratio of intracellular to extracellular cathepsin D consistent with endogenous IGF-II interfering with intracellular routing of lysosomal enzymes to lysosomes and inhibition of uptake of lysosomal enzymes from the media.